Apparatus for screening compound libraries

ABSTRACT

Disclosed are apparatus for screening compound libraries using frontal chromatography in combination with mass spectrometry to identify and rank those members of the library that bind to a target receptor. The apparatus of this invention also permit a compound library to be rapidly screened to determine if any member of the library has an affinity for the target receptor as measured by a pre-selected indicator compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.09/069,890, filed Apr. 29, 1998, which application claims the benefit ofU.S. Provisional Application No. 60/079,622, filed Mar. 27, 1998. Eachof these applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to apparatus for screening compoundlibraries, such as compound libraries generated using combinatorialchemistry techniques. The apparatus of this invention employ frontalchromatography in combination with mass spectrometry to screen a libraryof compounds to identify and rank those members of the library that bindto a target receptor. The apparatus of this invention also permit acompound library to be rapidly screened to determine if one or moremembers of the library have an affinity for a target receptor asmeasured by a pre-selected indicator compound.

[0004] 2. References

[0005] The following publications, patents and patent applications arecited in this application as superscript numbers:

[0006]¹ K. S. Lam, Anti-Cancer Drug Des. 1997, 12, 145-167.

[0007]² P. M. Sweetnam et al., In Burger's Medicinal Chemistry and DrugDiscovery; M. E. Wolff, Ed.; John Wiley & Sons: New York, 1995; pp697-731.

[0008]³ R. H. Griffey et al., In Proceedings of the 45^(th) ASMSConference on Mass Spectrometry and Allied Topics, Palm Springs, Calif.,Jun. 1-5, 1997; p. 400.

[0009]⁴ L. Fang et al., In Proceedings of the 45^(th) ASMS Conference onMass Spectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-5,1997; p. 401.

[0010]⁵ Y.-H. Chu et al., J. Am. Chem. Soc. 1996, 118, 7827-7835.

[0011]⁶ Y.-Z. Zhao et al., J. Med. Chem. 1997, 40, 4006-4012.

[0012]⁷ Y. F. Hsieh et al., J. Mol. Div. 1996, 2, 189-196.

[0013]⁸ R. W. Nelson et al., Anal. Chem. 1995, 67, 1153-1158.

[0014]⁹ D. C. Schriemer and L. Li, Anal. Chem. 1996, 68, 3382-3387.

[0015]¹⁰ PCT/US97/07964 (International Publication No. WO 97/43641),published Nov. 20, 1997, entitled “Molecular Diversity Screening Deviceand Method.”

[0016]¹¹ R. Wieboldt et al., Anal. Chem. 1997, 69, 1683-1691.

[0017]¹² R. B. van Breemen et al., Anal. Chem. 1997, 69, 2159-2164.

[0018]¹³ M. L. Nedved et al., Anal. Chem. 1996, 68, 4228-4236.

[0019]¹⁴ PCT/US95/03355 (International Publication No. WO 95/25737),published Sep. 28, 1995, entitled “Method for Identifying Members ofCombinatorial Libraries.”

[0020]¹⁵ PCT/EP97/02215 (International Publication No. WO 97/43301),published Nov. 20, 1997, entitled “Identification of Members ofCombinatorial Libraries By Mass Spectrometry.”

[0021] All of the above publications, patents and patent applicationsare herein incorporated by reference in their entirety to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety.

[0022] State of the Art

[0023] In recent years, a large number of combinatorial chemistrytechniques have been developed which permit vast libraries of diversechemical compounds to be rapidly synthesized.¹ In combinatorialchemistry, a series of chemical reactions is typically conductedemploying a plurality of reagents at each step to generate a library ofcompounds. Such techniques have the potential to greatly accelerate thediscovery of new compounds having biologically useful properties byproviding large collections of diverse chemical compounds for biologicalscreening.

[0024] This ability to rapidly generate large collections of compoundsusing combinatorial chemistry techniques has created a need for newmethods of screening compound libraries. The traditional approach ofscreening each compound individually in an assay to identify thosecompounds having the desired biological activity is no longer practicaldue to time and resource constraints. Thus, a need exists for newmethods and apparatus which permit the rapid screening of compoundlibraries.

[0025] In this regard, various methods for screening compound librarieshave been reported. Typically, these screening methods involve the useof target receptors which have been labeled with fluorescent or otherreporter groups.² In these methods, the compound library, typicallybound to a resin bead, is exposed to the labeled target receptor andthose members binding to the labeled target receptor are identified andphysically separated. The particular ligand binding to the targetreceptor is then identified. In many of these techniques, elaborateprocedures are required to keep track of individual members of thelibrary. For example, coded tags are often added during the synthesis ofthe combinatorial library to allow the structure of the individualmembers to be subsequently determined. Alternatively, combinatoriallibraries can be prepared in an array and the individual members of thelibrary subsequently identified by their location in the array. Whilesuch methods can be effective, the need to keep track of individualmembers of the library during their synthesis and screening is quitecumbersome and often limits the type of synthetic procedures that can beemployed. Additionally, many of these techniques require that thesynthetic procedures be conducted on a solid phase, thus furtherlimiting the synthetic procedures and reagents that can be used.

[0026] As an alternative, mass spectrometry is emerging as an importanttool for the interrogation of combinatorial libraries. To date, massspectrometry has been used to assess library quality^(3,4) and, whencoupled with molecular recognition technologies, has allowed for somesuccess in the isolation and characterization of active librarycompounds.⁵⁻¹⁵ Typically, when screening compound libraries forbiologically active members, mass spectrometry is used in combinationwith a “capture and release” methodology. In this methodology, compoundmixtures are presented to the target receptor, which is oftenimmobilized on a solid support, and the resulting ligand-receptorcomplexes are separated from the unbound members of the library. Afterseparation, the ligand-receptor complexes are typically denatured, forexample, with a solvent and the solvent mixture containing thepreviously bound ligands is presented to the mass spectrometer to permitidentification of the high affinity ligands.

[0027] For example, ultrafiltration has been used in combination withelectrospray mass spectrometry to screen combinatorial libraries.¹⁰⁻¹²In this method, ligands present in a compound library are allowed tobind to a receptor and the resulting ligand-receptor complexes arepurified by ultrafiltration. The ligand-receptor complexes are thendissociated using a solvent, such as methanol, and the previously boundligands are detected by an electrospray mass spectrometer.

[0028] Affinity capillary electrophoresis (ACE) has also been coupledwith mass spectrometry to screen combinatorial libraries.⁵ In thisprocedure, ACE is used to separate ligand-receptor complexes fromunbound ligands and mass spectrometry is used to identify the highaffinity ligands.

[0029] Similarly, compound libraries have been screened using affinitychromatography in combination with mass spectrometry. For example, WO97/43301 describes a method for characterizing the members of acombinatorial library, which method utilizes affinity selection incombination with mass spectrometry. Specifically, the members of thelibrary are brought into contact with a domain of interest to allow forbinding, i.e., the formation of a complex. After binding, the complex isseparated from the unbound members of the library, typically by washingthe unbound members from the column containing the complexes. Thecomplexes are then treated to elute the bound library components and theeluted components are analyzed by mass spectrometry. The elution methodsdescribed include the use of displacers, chaotrope agents, pH elution,salt gradients, temperature gradients, organic solvents, selectivedenaturants and detergents. Using such methods, the weakly bound membersof the library are purportedly eluted first and analyzed by massspectrometry, followed by the elution of the more strongly boundmembers.

[0030] There are several disadvantages associated with the “capture andrelease” methods for screening compound libraries that have beenpreviously reported. First, the procedure used to “release” the boundligands from the ligand-receptor complexes may alter the binding profilefor the various bound ligands, resulting in a false indication ofbinding strength. For example, using a pH gradient to release the boundmembers of the library may change the electronic character of thebinding site on the receptor causing ligands which are strongly boundunder physiological conditions to be prematurely released. Thus, thecharacterization of binding strength for various ligands based on theirrelative time of release may be misleading if the release conditions aredifferent from the binding conditions. Accordingly, these methods maynot accurately identify the most active members of a compound library.Additionally, certain conditions used for compound release, such as pHgradients, may irreversibly denature the receptor thus preventing itssubsequent use for screening compound libraries.

[0031] Additionally, when “capture and release” methods are employed,each bound ligand is typically released over a relatively short periodof time resulting, for example, in an elution peak or “spike” for eachligand. Accordingly, the effluent produced using such methods istypically monitored continually, for example, by mass spectrometry sothat any particular elution peak is not missed. Thus, the number ofanalyses that can be conducted using any particular mass spectrometer islimited. Accordingly, it would be desirable to develop methods andapparatus for screening compound libraries that do not rely upon“capture and release” methodologies.

SUMMARY OF THE INVENTION

[0032] This invention is directed to apparatus for screening compoundlibraries. The compound libraries may be generated or obtained by anymeans including, by way of example, combinatorial chemistry techniquesor from fermentation broths, plant extracts, cellular extracts and thelike. The apparatus of this invention employ frontal chromatography (FC)in combination with mass spectrometry (MS) to screen the library ofcompounds to identify and rank those members of the library that bind toa target receptor.

[0033] In frontal chromatography, a target receptor is typicallyimmobilized on a suitable solid support material and packed in a column.A mixture containing putative ligands is then continuously infusedthrough the column. Ligands having an affinity for the target receptorbind to the column, but eventually the capacity of the column for eachligand is exceeded and the ligands elute or “break through” at theirinfusion concentration. Once a ligand begins eluting from the column, itis continually present in the effluent. Compounds having little or noaffinity for the target receptor break through earlier in the effluentcompared to ligands having a higher affinity for the receptor.

[0034] In the present invention, mass spectrometry (MS) is employed tocontinuously or intermittently monitor the FC effluent. Using MS, theidentity and break through time for each ligand on the column can bedetermined. Thus, FC-MS allows the relative affinity of each member ofthe library for the target receptor to be determined relative to othermembers of the library under ligand-receptor binding conditions. Usingthe present apparatus, an accurate ranking of the relative affinity ofeach member of the compound library for the target receptor can beascertained.

[0035] Accordingly, in one of its apparatus aspects, the presentinvention is directed to an apparatus for screening a compound libraryto determine the relative or absolute affinity of a plurality ofputative ligands to a target receptor or a plurality of targetreceptors, which apparatus comprises:

[0036] (a) a column comprising a target receptor or a plurality oftarget receptors, each target receptor optionally attached to a solidphase support, and having a inflow end and an outflow end, wherein saidcolumn is capable of having a compound library comprising a plurality ofputative ligands applied thereto under frontal chromatography conditionsto produce an effluent from the outflow end of the column;

[0037] (b) a first reservoir connected to the inflow end of said columnfor applying the compound library to the column;

[0038] (c) a mass spectrometer connected to the outflow end of saidcolumn for continuously or intermittently analyzing the effluent fromthe column.

[0039] In a preferred embodiment, the above described apparatus furthercomprises:

[0040] (d) a second reservoir connected to the inflow end of the columnfor applying either (i) a mixture comprising the compound library, atleast one void marker compound and an indicator compound or a pluralityof indicators compounds, (ii) at least one void marker compound and anindicator compound or a plurality of indicator compounds, or (iii) abuffer solution to the column.

[0041] In another preferred embodiment, the above described apparatusfurther comprises:

[0042] (e) a third reservoir connected to the outflow end of the columnfor supplying a supplemental diluent to the effluent before analysis bythe mass spectrometer.

[0043] Preferably, the column employed in this invention will have aninternal diameter (i.d.) ranging from about 10 μm to about 4.6 mm. Morepreferably, the internal diameter of the column will be in the range offrom about 100 μm to about 250 μm.

[0044] Preferably, the column will range in length from about 1 cm toabout 30 cm, more preferably from about 2 cm to about 20 cm.

[0045] Preferably, each target receptor is independently selected fromthe group consisting of proteins, including recombinant proteins,glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes,lectins, selecting, cell-adhesion molecules, toxins, bacterial pili,transport proteins, receptors involved in signal transduction orhormone-binding, hormones, antibodies, major histocompatabilitycomplexes, immunoglobulin superfamilies, cadherins, DNA or DNAfragments, RNA and RNA fragments, whole cells, cell fragments, tissues,bacteria, fungi, viruses, parasites, preons, and synthetic analogs orderivatives thereof.

[0046] Additionally, each target receptor is preferably bound to a solidphase support. More preferably, each target receptor is covalently boundto the solid phase support or bound via biotin-avidin orbiotin-streptavidin binding.

[0047] Preferably, the solid phase support used in this invention isselected from the group consisting of polymeric (resin) beads, polymericgels, glass beads, silica chips, silica capillaries, agarose,diatomaceous earths and pulp.

[0048] The column employed in this invention preferably contains fromabout 1 fmol to about 10 nmol of target receptor active sites;preferably, from about 1 pmol to about 10 nmol of target receptor activesites.

[0049] Preferably, the mass spectrometer employed in this invention isan electrospray mass spectrometer.

[0050] Additionally, since FC-MS does not require constant effluentmonitoring, a plurality of FC-MS analyses can be conductedsimultaneously using a single mass spectrometer to intermittentlymonitor each column. Furthermore, under FC conditions, since ligands arealways present in the effluent once they break through the column, theintermittent monitoring of each column does not necessarily requiremonitoring the actual break through time for each ligand. Therefore, aplurality of FC-MS analyses can be conducted simultaneously using asingle mass spectrometer to intermittently monitor each column.

[0051] Accordingly, in another of its apparatus aspects, this inventionprovides an apparatus for screening a plurality of compound libraries todetermine the relative or absolute affinity of a plurality of putativeligands in each library to a target receptor or a plurality of targetreceptors, which apparatus comprises:

[0052] (a) a plurality of columns each column comprising a targetreceptor or a plurality of target receptors, each target receptoroptionally attached to a solid phase support, and each column having ainflow end and an outflow end, wherein each of said columns is capableof independently having a compound library comprising a plurality ofputative ligands applied thereto under frontal chromatography conditionsto produce an effluent from the outflow end of the column;

[0053] (b) a plurality of first reservoirs each connected to the inflowend of one of the columns for applying a compound library to thecolumns;

[0054] (c) a mass spectrometer connected to the outflow end of each ofsaid columns for intermittently analyzing the effluent from each of thecolumn.

[0055] In a preferred embodiment, the above described apparatus furthercomprises:

[0056] (d) a plurality of second reservoirs each connected to the inflowend of one of the columns for applying either (i) a mixture comprisingthe compound library, at least one void marker compound and an indicatorcompound or a plurality of indicator compounds, (ii) at least one voidmarker compound and an indicator compound or a plurality of indicatorcompounds, or (iii) a buffer solution to the column.

[0057] In another preferred embodiment, the above described apparatusfurther comprises:

[0058] (e) a third reservoir connected to the outflow end of each of thecolumns for supplying a supplemental diluent to the effluent from eachcolumn before analysis by the mass spectrometer.

[0059] Preferably, the above described apparatus comprises from 2 toabout 100 columns, more preferably from 3 to about 50 columns; and stillmore preferably from 5 to about 10 columns.

[0060] Preferably, each column is intermittently monitored for a periodof about 0.5 seconds to about 10 seconds, preferably for about 1 secondto about 5 seconds, before switching to the next column.

[0061] The apparatus of this invention can also be employed to screen atarget receptor or a plurality of target receptors for affinity to animmobilized ligand or plurality of ligands. This embodiment isparticularly useful for target validation studies on ligands havingbiological effects. Accordingly, in another of its apparatus aspects,this invention provides an apparatus for screening a target receptor ora plurality of target receptors to determine the relative affinity ofthe receptor or receptors to an immobilized ligand or ligands relativeto an indicator compound or a plurality of indicator compounds, whichapparatus comprises:

[0062] (a) a column comprising a ligand or a plurality of ligandswherein each ligand is bound to a solid phase support, said columnhaving a inflow end and an outflow end and further wherein said columnis capable of having a target receptor or a plurality of targetreceptors applied thereto under frontal chromatography conditions toproduce an effluent from the outflow end of the column;

[0063] (b) a first reservoir connected to the inflow end of said columnfor applying the target receptor or receptors to the column;

[0064] (c) a second reservoir connected to the inflow end of the columnfor applying either (i) a mixture comprising the target receptor orreceptors, at least one void marker compound and an indicator compoundor a plurality of indicators compounds, (ii) at least one void markercompound and an indicator compound or a plurality of indicatorcompounds, or (iii) a buffer solution to the column.

[0065] (d) a mass spectrometer connected to the outflow end of saidcolumn for continuously or intermittently analyzing the effluent fromthe column.

[0066] In a preferred embodiment, the above apparatus further comprises:

[0067] (e) a third reservoir connected to the outflow end of the columnfor supplying a supplemental diluent to the effluent before analysis bythe mass spectrometer.

[0068] In a preferred embodiment, each ligand employed in the aboveapparatus is selected from the group consisting of carbohydrates,monosaccharides, oligosaccharides, polysaccharides, amino acids,peptides, oligopeptides, polypeptides, proteins, nucleosides,nucleotides, oligonucleotides, polynucleotides, lipids, retinoids,steroids, glycopeptides, glycoproteins, glycolipids, proteoglycans, andsynthetic analogs or derivatives thereof.

[0069] In another preferred embodiment, each ligand is selected from thegroup consisting of synthetic small molecule organic compounds.

[0070] A plurality of such FC-MS analyses can also be conductedsimultaneously using a single mass spectrometer to intermittentlymonitor each column. Accordingly, in yet another of its apparatusaspects, the present invention provides an apparatus for screening aplurality of target receptors to determine the relative affinity of thereceptors to an immobilized ligand or ligands relative to an indicatorcompound or a plurality of indicator compounds, which apparatuscomprises:

[0071] (a) a plurality of columns each column comprising a ligand or aplurality of ligands wherein each ligand is bound to a solid phasesupport, and each column having a inflow end and an outflow end, whereineach of said columns is capable of independently having a targetreceptor or a plurality of target receptors applied thereto underfrontal chromatography conditions to produce an effluent from theoutflow end of the column;

[0072] (b) a plurality of first reservoirs each connected to the inflowend of one of the columns for applying a target receptor or a pluralityof target receptors to the columns;

[0073] (c) a plurality of second reservoirs each connected to the inflowend of one of the columns for applying either (i) a mixture comprisingthe target receptor or plurality of target receptors, at least one voidmarker compound and an indicator compound or a plurality of indicatorcompounds, (ii) at least one void marker compound and an indicatorcompound or a plurality of indicator compounds, or (iii) a buffersolution to the column.

[0074] (d) a mass spectrometer connected to the outflow end of each ofsaid columns for intermittently analyzing the effluent from each of thecolumn.

[0075] In a preferred embodiment, the above apparatus further comprises:

[0076] (e) a third reservoir connected to the outflow end of each of thecolumns for supplying a supplemental diluent to the effluent from eachcolumn before analysis by the mass spectrometer.

[0077] Preferably, the above described apparatus comprises from 2 toabout 100 columns, more preferably from 3 to about 50 columns; and stillmore preferably from 5 to about 10 columns.

[0078] Preferably, each column is intermittently monitored for a periodof about 0.5 seconds to about 10 seconds, preferably for about 1 secondto about 5 seconds, before switching to the next column.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079]FIG. 1 illustrates a representative apparatus for screeningcompound libraries using frontal chromatography in combination with amass spectrometer.

[0080]FIG. 2 illustrates a representative apparatus for screeningcompound libraries using a plurality of frontal chromatography columnsin combination with a mass spectrometer.

[0081]FIG. 3 illustrates another representative apparatus for screeningcompound libraries using a plurality of frontal chromatography columnsin combination with a mass spectrometer.

[0082]FIG. 4 illustrates a representative apparatus for sequentiallyscreening compound libraries with an indicator compound using aplurality of frontal chromatography columns in combination with a massspectrometer.

[0083]FIG. 5A shows a total ion chromatogram (TIC) from a FC-MS runusing six representative oligosaccharides having varying affinity for acarbohydrate-binding antibody that recognizes the3,6-dideoxy-D-galactose (abequose) epitope in Salmonella paratyphi BO-antigens.

[0084]FIG. 5B shows selected ion chromatograms for the sixoligosaccharides reconstructed from the TIC shown in FIG. 5A.

[0085]FIGS. 5C, 5D and 5E show mass spectra generated from time-slicesof the TIC shown in FIG. 5A.

[0086]FIG. 6 shows a plot of ([A]₀(V-V₀))⁻¹ versus [A]₀ ⁻¹ forαGal(1→2)[αAbe(1→3)]αMan-OCH₃.

[0087]FIG. 7A shows a selected ion chromatogram from a FC-MS run usingan indicator compound in the absence of a compound library.

[0088]FIG. 7B shows a selected ion chromatogram from a FC-MS run usingan indicator compound in the presence of a compound library.

[0089]FIG. 8 shows a selected ion chromatogram from a FC-MS run usingfour representative oligosaccharides having varying affinity for choleratoxin B subunit.

[0090]FIG. 9 shows a selected ion chromatogram from a FC-MS run using asynthetically prepared GM₁ analog.

[0091]FIG. 10 illustrates the “roll-up” effect in a selected ionchromatogram from a FC-MS run using an indicator compound in thepresence of a compound library.

[0092]FIG. 11 is a graph of the reduction of column activity as afunction of time for two different compound libraries, the first librarycontaining many weak binders and the second library containing strongbinders.

[0093]FIG. 12A shows schematically the synthesis of a compound librarycontaining 100 tripeptides. FIG. 12B shows an electrospray mass spectrumof the compound library.

[0094]FIG. 13 shows a chromatogram of three infusion/wash cycles of acompound library containing 100 tripeptides, an indicator compound(dashed line) and a void marker compound (solid line).

[0095]FIG. 14A illustrates the V-V₀ value for an indicator compound(dashed line) relative to a void marker compound (solid line)immediately before equilibration of a column with a compound librarycontaining 100 tripeptides. FIG. 14B illustrates the V-V₀ value for theindicator compound immediately after equilibration of the column withthe compound library.

[0096]FIG. 15A shows a total ion chromatogram for a compound librarycontaining 100 tripeptides. FIG. 15B shows a selected ion chromatogramfor m/z 419.2.

[0097]FIG. 16A shows a selected ion chromatogram for fPR. FIG. 16B showsa selected ion chromatogram of fPR and fPR-chloromethyl ketone.

DETAILED DESCRIPTION OF THE INVENTION

[0098] The present invention provides apparatus for screening compoundlibraries using frontal chromatography in combination with massspectrometry. When describing the apparatus of this invention, thefollowing terms have the following meanings, unless otherwise indicated.All terms not defined herein have their conventional art-recognizedmeaning.

[0099] Definitions

[0100] The term “buffer” refers to a solution that stabilizes thebinding activity of the target receptor. Typical buffers include, by wayof illustration, pH buffers and buffers containing specific molecules,either organic or inorganic, required to stabilize the binding activityof a specific target receptor.

[0101] The term “break through time” refers to the period of timebetween elution of the void volume and the front corresponding to theelution of a particular compound during frontal chromatography. The term“break through curve” refers to the signal intensity as a function oftime resulting from the infusion of compound(s) through a column underfrontal chromatography conditions. Typically, the break through curve iscomprised of a front (fast-rising section) and a plateau (horizontalsection).

[0102] The term “compound library” refers to a mixture or collection ofone or more putative ligands generated or obtained in any manner.Preferably, the library contains more than one putative ligand ormember.

[0103] The term “electrospray” refers to the generation of gas-phaseions from a flowing solution. Electrospray is typically performed atatmospheric pressure in an electric field with or without assistednebulization and solvent evaporation.

[0104] The term “effluent” refers to the solvent or solution emerging orexiting from the frontal chromatography column.

[0105] The term “frontal chromatography conditions” refers tochromatography conditions in which a solution of compounds, such asputative ligands and/or indicator compounds, is applied or infusedthrough a column to generate a break through curve. Typically, underfrontal chromatography conditions, putative ligands are infusedcontinuously at a constant concentration through a column containing atarget receptor such that the target receptor is continuously contactedwith the putative ligands during the chromatography.

[0106] The term “indicator compound” or “indicator” refers to a compoundhaving a known affinity or specificity for the target receptor and ameasurable break through time under frontal chromatography conditions.When screening target receptors for affinity to a particular ligand(s),the indicator may also be a compound, such as a protein, or otherbiological entity, such as a cell or cell fragment, having a knownaffinity or specificity for the ligand(s). The break through time forthe indicator is typically referenced to the void volume of the system.

[0107] The term “ligand” refers to a molecule or group of molecules thatbind to one or more specific sites of a receptor. Representative ligandsinclude, by way of illustration, carbohydrates, monosaccharides,oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides,polypeptides, proteins, nucleosides, nucleotides, oligonucleotides,polynucleotides, including DNA and DNA fragments, RNA and RNA fragmentsand the like, lipids, retinoids, steroids, glycopeptides, glycoproteins,glycolipids, proteoglycans and the like, and synthetic analogues orderivatives thereof, including peptidomimetics, natural products ornaturally-occurring small molecule organic compounds (i.e., compoundsproduced by and/or isolated from natural sources, such as soil, water,cells, plants, fungi, animals and the like), synthetic small moleculeorganic compounds, inorganic ions, organometallic compounds and thelike, and mixtures thereof. The term “putative ligand” refers to aligand whose affinity or specificity for a target receptor, if any, hasnot been determined.

[0108] The term “microcolumn” refers to a column having an internaldiameter less than or equal to about 1 mm.

[0109] The term “natural products” refers to compounds isolated fromnatural sources, such as cells, plants, fungi, animals and the like. Theterm “naturally-occurring small molecule organic compounds” refers tonatural products that are organic compounds generally having a molecularweight less than about 1000, preferably less than about 500.

[0110] The term “selected ion chromatogram” refers to a plot of ionabundance vs. time constructed from the intensity of a single ion. Aselected ion chromatogram can be prepared from a scan or selected ionmonitoring mode.

[0111] The term “selected ion monitoring” refers to the detection of afew pre-selected ions using a mass spectrometer (e.g. quadrupoles).

[0112] The term “signal intensity” refers to the measured response of aninstrument to a stimulus, for example, the current output of a highenergy dynode detector resulting from the impact of an ion.

[0113] The term “solid support” or “solid phase support” refers to aninert material or molecule to which a target receptor may be bound orcoupled, either directly or through a linking arm.

[0114] The term “synthetic small molecule organic compounds” refers toorganic compounds generally having a molecular weight less than about1000, preferably less than about 500, which are prepared by syntheticorganic techniques, such as by combinatorial chemistry techniques.

[0115] The term “supplemental diluent” or “make-up flow” refers to asolution or solvent which is combined with the effluent from a columnbefore the effluent passes through the mass analyzer of a massspectrometer.

[0116] The term “target receptor” or “receptor” refers to a molecule ora group of molecules capable of binding a ligand at a specific site.Representative examples of target receptors include, by way of example,proteins, including recombinant proteins, glycoproteins,glycosaminoglycans, proteoglycans, integrins, enzymes, lectins,selecting, cell-adhesion molecules, toxins, bacterial pili, transportproteins, receptors involved in signal transduction or hormone-binding,hormones, antibodies, major histocompatability complexes, immunoglobulinsuperfamilies, cadherins, DNA or DNA fragments, RNA and RNA fragments,whole cells, cell fragments, tissues, bacteria, fungi, viruses,parasites, preons, and synthetic analogs or derivatives thereof.

[0117] The term “target receptor active site” refers to the binding siteof interest on a particular target receptor.

[0118] The term “total ion chromatogram” refers to a plot of ionabundance vs. time constructed from a summation of all ion intensitiesin a scan. In a total ion chromatogram, the number of scans are linearlyrelated to time.

[0119] The term “void marker compound” or “void marker” refers to acompound that elutes from column at the void volume. The void markercompound is used to identify the void volume of a column used underfrontal chromatography conditions.

[0120] The term “void volume” or “V₀” refers to the volume of solutionwhich passes through a frontal chromatography column from the point ofinfusion to the point of detection of a compound, i.e. a putativeligand, in the absence (or suppression) of a binding event. Since theflow rate is typically constant, void volume is generally specified interms of a retention time for the compound. Putative ligands having noaffinity for the target receptor typically elute from column at the voidvolume.

[0121] The compound libraries employed in this invention may be preparedor obtained by any means including, but not limited to, combinatorialchemistry techniques, fermentation methods, plant and cellularextraction procedures and the like. Methods for making combinatoriallibraries are well-known in the art. See, for example, E. R. Felder,Chimia 1994, 48, 512-541; Gallop et al., J. Med. Chem. 1994, 37,1233-1251; R. A. Houghten, Trends Genet. 1993, 9, 235-239; Houghten etal., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84;Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectivesin Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992,89, 5381-5383; Gordon et al., J. Med. Chem. 1994, 37, 1385-1401; Lebl etal., Biopolymers 1995, 37 177-198; and references cited therein. Each ofthese references is incorporated herein by reference in its entirety.

[0122] Any type of molecule that is capable of binding to a targetreceptor may be present in the compound library. For example, compoundlibraries screened using this invention may contain naturally-occurringmolecules, such as carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, peptides, oligopeptides, polypeptides,proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides,including DNA and DNA fragments, RNA and RNA fragments and the like,lipids, retinoids, steroids, glycopeptides, glycoproteins, glycolipids,proteoglycans and the like; or analogs or derivatives ofnaturally-occurring molecules, such peptidomimetics and the like; andnon-naturally occurring molecules, such as “small molecule” organiccompounds generated, for example, using combinatorial chemistrytechniques; organometallic compounds, inorganic ions, and mixturesthereof. The term “small molecule organic compound” refers to organiccompounds generally having a molecular weight less than about 1000,preferably less than about 500.

[0123] A particular advantage of FC-MS is that compound librariescontaining isomers may be screened to determine, for example, if onlyone isomer (e.g. an enantiomer or diastereomer) is binding to the targetreceptor, or if the isomers have different affinities for the targetreceptor. In this regard, if the isomers have different affinities forthe target receptor, a different break through time will be observed foreach isomer.

[0124] The compound libraries employed in this invention will typicallycontain a plurality of members or putative ligands. When a indicatorcompound is employed, the compound library will preferably contain lessthan about 50,000 members, more preferably, the compound library willcontain less than about 10,000 members. When an indicator compound isnot employed, the compound library will preferably contain less thanabout 10,000 members; more preferably, from 1 to about 1,000 members;and still more preferably, from about 5 to about 100 members.

[0125] The present apparatus is useful for analyzing the affinity ofmembers of a compound library for any target receptor or domain whichbinds or complexes with a ligand. For example, the target receptor maybe selected from, but is not limited to, proteins, including recombinantproteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins,enzymes, lectins, selectins, cell-adhesion molecules, toxins, bacterialpili, transport proteins, receptors involved in signal transduction orhormone-binding, hormones, antibodies, major histocompatabilitycomplexes, immunoglobulin superfamilies, cadherins, DNA or DNAfragments, RNA and RNA fragments, whole cells, cell fragments, tissues,bacteria, fungi, viruses, parasites, preons, and synthetic analogs orderivatives of any of the above. If desired, more than one targetreceptor may be employed when screening a compound library using themethods of this invention.

[0126] When employing the apparatus of this invention, the targetreceptor (or a ligand) is optionally bound or coupled to a solidsupport. Preferably, the target receptor is covalently bound or coupledto the solid support. However, in some cases, such as when whole cellsor organisms are employed as the target receptor, the cells or organismsmay be contained within the column by using, for example, a porous fritor membrane at the outflow end of the column. Supports for receptors arewell-known in the art and many are commercially available. Any suchconventional support may be used in this invention. Representativesupports include, by way of illustration, polymeric (resin) beads,polymeric gels, glass beads, silica chips and capillaries, agarose,diatomaceous earths, pulp, and the like. When silica capillaries areused as the solid support, the target receptor is bound directly to thewalls of the column. Preferred solid supports for use in this inventionare those having minimal non-specific binding properties. A preferredsolid support is derivatized porous polystyrene-divinylbenzene polymerbeads, such as POROS beads (available from Perseptive Biosystems,Framingham, Mass.). A particularly preferred solid support is silicaparticles, such as controlled pore glass (available from CPG Inc.,Lincoln Park, N.J.).

[0127] The target receptor (or ligand) can be bound or coupled to thesupport using any art-recognized procedure. For example, the targetreceptor can be bound using direct immobilization techniques (i.e.,covalent binding via a sulfhydryl, amino or carboxyl group and thelike), covalent binding through a linking or spacer arm, biotin-avidinbinding, biotin-streptavidin binding, antibody binding such asantibody-protein A binding, GST-glutathione binding, ion exchangeabsorption, hydrophobic interaction, expression of the target receptoras a recombinant protein fused to maltose binding protein, fusion of thetarget receptor with a peptide which binds selectively to an affinitycolumn, and the like. Such methods are well-known in the art and kitsfor practicing many of these methods are commercially available. See,for example, Stammers et al.., FEBS Lett. 1991, 283, 298-302; Herman etal.., Anal. Biochemistry 1986, 156, 48; Smith et al., FEBS Lett. 1987,215, 305; Kilmartin et al., J. Cell. Biol. 1982, 93, 576-582; Skinner etal., J. Biol. Chem. 1991, 266, 14163-14166; Hopp et al., Bio/Technology1988, 6, 1204-1210; H. M. Sassenfeld, TIBTECH 1990, 8, 88-93; Hanke etal., J. General Virology 1992, 73, 654-660; Ellison et al., J. Biol.Chem. 1991, 267, 21150-21157; U. K. Pati, Gene 1992, 114, 285-288;Wadzinski et al., J. Biol Chem. 1992, 267, 16883-16888; Field et al.,Mol. Cell. Biol. 1988, 8, 2159-2165; Gerard et al., Biochemistry 1990,29, 9274-9281; Ausselbergs et al., Fibrinolysis 1993, 7, 1-13; Hopp etal., Biotechnology 1988, 6, 1205-1210; Blanar et al., Science 1992, 256,1014-1018; Lin et al., J. Org. Chem. 1991, 56, 6850-6856; Zastrow etal., J. Biol. Chem. 1992, 267, 3530-3538; Lim et al., J. InfectiousDisease 1990, 162, 1263-1269; Goldstein et al., Virology 1992, 190,889-893; and the articles in IBI FLAG Epitope Vol. 1: No. 1, September1992; and references cited therein. Each of these references isincorporated herein by reference in its entirety.

[0128] In a preferred embodiment of this invention, the target receptoris bound or coupled to the solid support using biotin-avidin,biotin-streptavidin or a related-type binding. In this procedure, thetarget receptor is typically biotinylated with a biotin reagentcontaining a spacer arm. The biotinylated target receptor is thencontacted with an avidin-containing solid support. The resultingbiotin-avidin complex binds the target receptor to the solid support.

[0129] Procedures for biotinylating biomolecules are well-known in theart and various biotin reagents are commercially available. See, forexample, E. A. Bayer et al., Meth. Enzymol. 1990, 184, 51; U. Bickel etal., Bioconj. Chem. 1995, 6, 211; H. Hagiwara et al., J. Chromatog.1992, 597, 331; “Avidin-Biotin Chemistry Handbook” (available fromPierce, Rockford, Ill., Catalog Item No. 15055) and references citedtherein. A preferred biotin reagent is NHS-LC-biotin (available fromPierce). The extent of biotin incorporation using such reagents can bemonitored by, for example, matrix-assisted laser desorption/ionizationas described in D. C. Schriemer and L. Li, Anal. Chem. 1996, 68,3382-3387, or by other art-recognized methods as described in the“Avidin-Biotin Chemistry Handbook” (Pierce). Preferably, an average ofabout 1 to about 50 biotins are incorporated per target receptor, morepreferably about 1 to about 10 biotins per target receptor.

[0130] The biotinylated target receptor is typically coupled with anavidin- or streptavidin-containing solid support or related material.Such supports are commercially available or can be prepared byart-recognized procedures. Preferred avidin-containing supports includeUltralink-immobilized avidin (available from Pierce) and POROS 20immobilized streptavidin (available from Perseptive Biosystems). Thebiotinylated target receptor is typically coupled with theavidin-containing support by contacting the receptor with the support ina suitable buffer, such as phosphate buffered saline (pH 7), for about0.5 to 4 hours at a temperature ranging from about 4° C. to about 37° C.Preferably, after coupling the biotinylated target receptor to theavidin-containing support, any remaining avidin binding sites on thesupport are blocked by contacting the solid support with an excess offree biotin.

[0131] The target receptor may be bound or coupled to the solid supporteither prior to or after introducing the solid support material into acolumn. For example, the biotinylated target receptor may be contactedor incubated with the avidin- or streptavidin-containing solid supportand the resulting solid support containing the target receptorsubsequently introduced into a column. Alternatively, the avidin- orstreptavidin-containing solid support can be first introduced into thecolumn and the biotinylated target receptor then cycled through thecolumn to form the solid support containing the target receptor in thecolumn. Either of these methods may also be used with any of the otherpreviously mentioned procedures for coupling the target receptor to thesolid support.

[0132] When more than one target receptor is employed, each targetreceptor can be bound to the same solid support using the proceduresdescribed herein. Alternatively, each target receptor can be bound to aseparate solid support and the solid support materials containing thetarget receptors subsequently homogenized and packed into the column.

[0133] The solid support material may be introduced into the columnusing any conventional procedure. Typically, the solid support isslurried in a suitable diluent and the resulting slurry is pressurepacked or pumped into the column. Suitable diluents include, by way ofexample, buffers such as phosphate buffered saline (PBS) solutions,preferably containing a preservative such as sodium azide, and the like.

[0134] Generally, the activity of the target receptor will determine thesize of the column employed in this invention, i.e., a smaller columnvolume may be employed when the target receptor has more activity perunit column volume. Typically, the column employed in this inventionwill have an internal diameter (i.d.) ranging from about 10 μm to about4.6 mm. Preferably, the internal diameter of the column will be in therange of from about 100 μm to about 250 μm. The column will typicallyrange in length from about 1 cm to about 30 cm, preferably from about 2cm to about 20 cm. Preferably, the column will have from about 1 fmol toabout 10 nmol of target receptor active sites per column; morepreferably, from about 0.1 pmol to about 10 nmol of target receptoractive sites per column; still more preferably, from about 0.1 pmol toabout 100 pmol of target receptor active sites per column.

[0135] If an indicator compound is employed, the length of the columnand its i.d. will also depend upon the K_(d) of the indicator compound(i.e., a smaller column may be used when the indicator has a higheraffinity for the target receptor). Preferably, when an indicator isemployed, the column length and i.d. are selected so that the indicatorcompound elutes a measurable quantity after the void volume.

[0136] The body of the column employed in this invention may becomprised of any conventional column body material including, by way ofillustration, poly(ether ether ketone) (PEEK), fused silica, siliconmicrochips, stainless steel, nylon, polyethylene,polytetrafluoroethylene (Teflon) and the like. Preferably, the columnbody is comprised of poly(ether ether ketone).

[0137] Alternatively, the column may be open-faced, such as inthin-layer chromatography (TLC) plate configuration or a gel plate. Inthis embodiment, the effluent can be analyzed using the electrospraytechniques described herein. Alternatively, the plate or plate-likeconfiguration can be subjected to matrix-assisted laserdesorption/ionization (MALDI) analysis at any stage of thechromatography to provide a molecular weight analysis for a plurality ofpositions on the plate.

[0138] After the solid support containing the target receptor isintroduced or formed in the column, the column is typically flushed witha suitable diluent to remove any unbound target receptor or impurities.Suitable diluents for flushing the column include, for example,phosphate buffered saline, TRIS buffers and the like. If desired, adetergent may also be included in the buffer to facilitate removal ofunbound target receptor or impurities.

[0139] After the column is flushed, the column is typically equilibratedwith a buffer suitable for frontal chromatography and compatible withmass spectrometry. Volatile buffers are generally preferred for use withmass spectrometry. For frontal chromatography, a buffer is typicallyselected to promote receptor-ligand interaction. Suitable buffers foruse in FC-MS include, by way of example, ammonium acetate, ammoniumformate and the like.

[0140] Following equilibration of the column, the compound library isthen applied to the column under frontal chromatography conditions.Typically, when applied to the column, the compound library comprises asolution of the library members or putative ligands in a suitablediluent. Typically, the diluent is the buffer solution used toequilibrate the column. Generally, the concentration of the librarymembers in the diluent will range from about 1 pM to about 50 μM.Preferably, the concentration of library members ranges from about 1 nMto about 10 μM.

[0141] Procedures for conducting frontal chromatography are well-knownin the art. See, for example, K.-I. Kasai et al., Journal ofChromatography 1986, 376, 33-47; D. S. Hage et al., Journal ofChromatography B, 1997, 669, 449-525 and references cited therein. Thedisclosures of these references are incorporated herein by reference intheir entirety. Typically, the compound library is continuously appliedor infused into the column containing the target receptor. Under theseconditions, the target receptor is continuously contacted or challengedwith each of the members of the compound library. The column is drivento dynamic equilibrium by continuously applying the compound library tothe column. Library members having different binding constants to thetarget receptor display different break through times or hold-up volumeson the column, i.e., those members having a higher affinity for thetarget ligand have a longer break through time on the column or a largerhold-up volume until they begin to elute from or break-though the columnat their initial infusion concentration. Unlike zonal chromatographicmethods, no physical separation of the library members is achieved usingfrontal chromatography. Suitable methods for conducting FC-MS aredescribed in U.S. patent application No. ________, filed on Dec. 28,1998 (which application is a continuation of U.S. Ser. No. 09/070,131,filed Apr. 29, 1998, now abandoned) and in U.S. Pat. No. ______, filedon even date herewith, as Attorney Docket No. 026579-250 and entitled“Methods for Screening Compound Libraries,” the disclosure of which areincorporated herein by reference in their entirety

[0142] During the frontal chromatography, the column is typically at atemperature in range from about 0° C. to about 90° C.; preferably fromabout 4° C. to about 60° C.; more preferably from about 20° C. to about40 ° C.

[0143] When a ligand has a very slow on-rate, it may be desirable toconduct the column equilibrium over an extended period of time. Thecolumn can be equilibrated by infusing the compound library through thecolumn for a period sufficient to allow the column to reach equilibrium.For example, this can be achieved by increasing the equilibration time,i.e., to about 0.25 to 24 hours; or by reducing the flow rate to about1% to 10% of the usual flow rate. Alternatively, a sequence of stop-flowcycles may also be conducted.

[0144] In the apparatus of this invention, a mass spectrometer iscoupled to the column to analyze the effluent. Mass spectrometry isparticularly useful in the present invention since it allows for bothdetection and identification of the library members present in theeffluent. In this regard, mass spectrometry allows the eluting membersof the library to be identified based on their mass/charge ratio.

[0145] Prior to analyzing the effluent from the column by massspectrometry, the effluent is optionally diluted with a supplementaldiluent or “make-up flow” and the combined flow is directed into, forexample, the electrospray mass spectrometer. Typically, the supplementaldiluent comprises a major amount of an organic solvent and a minoramount of an aqueous buffer. The organic solvent is selected so as topromote a stable and efficient electrospray. Representative organicsolvents suitable for use in the supplemental diluent include, by way ofexample, acetonitrile, methanol, isopropanol and the like. A preferredorganic solvent is acetonitrile. Typically, the amount of supplementaldiluent employed is adjusted so that the combined flow rate of theeffluent and the supplemental diluent is less than about 100 μL/min.Preferably, the combined flow rate entering the mass spectrometer rangesfrom about 100 nL/min to about 20 μL/min.

[0146] Methods for analyzing effluents using mass spectrometry arewell-known in the art. Any type of mass spectrometry which is capable ofdirectly or indirectly analyzing the components present in a solutionmay be employed in this invention including, for example, electrospraymass spectrometry (ES-MS), atmospheric pressure chemical ionization(APCI), membrane introduction mass spectrometry (MIMS), continuous flowfast atom bombardment (cf-FAB), thermospray techniques, particle beam,moving belt interfaces and the like. Electrospray mass spectrometry isparticularly preferred. Apparatus and techniques for conductingelectrospray mass spectrometric analysis are described, for example, inS. J. Gaskell, “Electrospray: Principles and Practice”, J. Mass.Spectrom. 1997, 32, 677-688 and reference cited therein. When theeffluent is collected and optionally pre-treated prior to mass spectralanalysis, any of the above described ionization methods may be used aswell as MALDI, fast atom bombardment (FAB), massive cluster impact,electron impact, chemical ionization, secondary ion mass spec and fielddesorption ionization techniques.

[0147] The mass spectrometer employed in the methods of this inventionmay be of any type (i.e., scanning or dynamic) including, by way ofillustration, quadrupole, time of flight, ion trap, FTICR and the like.Typically, the mass spectrometer parameters are set to provide thehighest sensitivity for the eluting compounds. Generally, when anelectrospray mass spectrometer is employed, such adjustments willinvolve optimization of, for example, nebulizer pressure, drying gasflow rate, ion transmission and electrospray needle position. Forexample, the nebulizer pressure will typically range from about 0 psi toabout 60 psi; and the drying gas flow rate will range from about 0 L/minto about 50 L/min. A total ion chromatogram is typically measured andmonitored in real-time. The size of the column, the concentration of thecompound library and the flow rate will generally determine therun-time. Typical run times range from about 1 min to about 60 min.

[0148] Upon completion of the frontal chromatography, the column isoptionally regenerated by washing with a large volume of the bindingbuffer, with or without a competitive ligand. In this regard, aparticular advantage of the present method is that denaturing of thetarget receptor is not required at any point in the procedure.Accordingly, columns may be re-used many times generally with noobservable loss of activity or leaching of the target receptor.Alternatively, since the methods of this invention employ very smallamounts of target receptor, the column may be disposed of after a singleuse.

[0149] A representative apparatus for conducting the screening methodsof this invention is illustrated in FIG. 1. As shown in FIG. 1, a firstreservoir 1, containing a buffer solution, and a second reservoir 2,containing a solution of a compound library in a buffer, are connectedvia tubing 3 to valve 4. In FIG. 1, reservoirs 1 and 2 are syringesalthough any similar reservoir may be employed. Valve 4 allows thesolutions from reservoirs 1 or 2 to be directed into a waste container 5or into the inflow end of column 6. Column 6 contains the targetreceptor bound to a solid phase support, the column wall or otherwiseretained within the column. The outflow end of column 6 is connected toa mixing tee 7, which is also connected to reservoir 8, containing asupplemental diluent, via tubing 9. The effluent from column 6 is mixedwith the supplemental diluent from reservoir 8 in mixing tee 7 and theoutflow is directed via tubing 10 to an electrospray mass spectrometer11. To control the flow from reservoirs 1, 2 and 8, pressure is appliedto plungers 12 via, for example, a pump. A simpler manifestation wouldinclude just the column 6 connected to the reservoir 2, with the outflowdirected via tubing 10 to an electrospray mass spectrometer.Alternatively, a configuration involving an HPLC pump and a valve withan oversized injection loop for the library solution could be used, suchan apparatus is described, for example, in E. Breklan et al., Biochem.1996, 35, 12141-12145.

[0150] In another of its embodiments, the apparatus of this inventioncan be used for screening a compound library to determine if any memberof the library has an affinity for a target receptor that interfereswith the binding of a pre-selected indicator compound or a mixture ofindicator compounds. In this embodiment, the break through time of anindicator compound having a known affinity for the target receptor isdetermined after the column has been equilibrated with the compoundlibrary and compared to the break through time for the indicatorcompound in the absence of the compound library. If the indicatorcompound has a shorter break through time after equilibration with thecompound library, the compound library contains one or more ligandshaving an overall affinity for the target ligand which is higher thanthe indicator compound. Since an indicator compound can be selectedhaving a relatively short break through time on the column, asignificant advantage of this embodiment is that compound libraries canbe rapidly screened, e.g., in less than 5 minutes, to identify thoselibraries having a pre-determined minimum level of affinity for thetarget receptor. When a library is identified as having thepre-determined minimum level of affinity for the target receptor, thelibrary can be further analyzed using FC-MS to identify the ligandsbinding to the target receptor.

[0151] One advantage of using an indicator compound is that thescreening time for each library is significantly reduced since only theindicator compound needs to be monitored relative to a void markercompound. Additionally, since the indicator compound binds to the targetreceptor at the active site of interest, a change in the break throughtime for the indicator reflects an affective interaction of a member (ormembers) of the library with the target receptor. This interactionincludes, by way of example, binding at the active site, and binding ata different , non-overlapping site that affects the ability of thetarget receptor to bind the indicator compound. This method isparticularly advantageous in that nonspecific binding to the targetreceptor that does not alter the active site will not cause a shift inthe break through time for the indicator compound. Accordingly,non-specific binding of the library to the target receptor does notprovide false leads.

[0152] The indicator compound used in this embodiment of the inventionis typically selected so as to have a relatively weak affinity for thetarget receptor. This permits the indicator compound to rapidly elute orbreak through the column, thus shortening the period of time necessaryto monitor the effluent. An indicator compound having a break throughtime on the column less than about 5 minutes in the absence of thecompound library is preferred. Alternatively, an indicator having astrong affinity for the target receptor may be used thereby allowingsmaller columns to be employed. When an indicator compound having astrong affinity is used, the compound library will typically be appliedto the column at a higher concentration. The break through time for theindicator compound on the column in the absence of the compound libraryis determined using the FC-MS procedures described herein. The affinityof the indicator compound for the target receptor can be determinedusing conventional techniques, such as microcalorimetry and the like; orby using the FC-MS methods of this invention. Preferably, the indicatorcompound will also have a unique mass in comparison to the members ofthe compound library so that the indicator compound can be unambiguouslyidentified by mass spectrometry. Generally, when using an indicatorcompound and a quadrupole mass spectrometer, only the m/z of theindicator compound and the compounds representing the void volume aremonitored to provide for a greater signal to noise ratio.

[0153] Representative examples of indicator compounds suitable for usewith specific target receptors include, by way of illustration,αAbe(1→3)αTal-OCH₃ (K_(d) =0.2 mM) for use with a monoclonal antibodythat recognizes the 3,6-dideoxy-D-galactose (abequose) epitope inSalmonella paratyphi B O-antigens; phytic acid (K_(d)=1 μM) for use withL-selectin, and the like. Additionally, more than one indicator compoundmay be employed. The indicator may also be coupled or conjugated toanother molecule containing an atom, isotope or molecular fragment whichfacilitates its detection. For example, the indicator compound can beconjugated to polyethylene glycols (PEGs) so that the mass spectra wouldcontain peaks differing by 44 units thereby facilitating detection ofthe of indicator compound.

[0154] The break through time for the indicator compound is typicallymeasured relative to a void marker compound. The void marker compound isa compound which elutes from the column at the void volume. Preferably,the void marker compound is structurally similar to the indicatorcompound, but has no affinity for the target receptor of interest. Insome cases, putative ligands in the compound library which have noaffinity for the target receptor may serve as the void marker compounds.

[0155] When a functional target receptor, such as an enzyme, is employedin this invention, the substrate for the functional target receptor maybe used as the indicator. By doing so, the loss of function orinhibition of the target receptor can be monitored in the presence of acompound library. In this embodiment, a first indicator compound isselected which is a substrate for the functional target receptor, i.e.the first indicator is a compound which is capable of being chemicallymodified by the functional target receptor to produce a second indicatorcompound. Using the frontal chromatography procedures described herein,the first indicator compound and the compound library to be analyzed areapplied to or infused into a column comprising the functional targetreceptor. The effluent from the column is then monitored for thepresence and/or concentration of the first and/or the second indicatorcompounds, i.e., the substrate and/or the reaction product. An increasein the expected concentration of the first indicator compound or adecrease in the expected concentration of the second indicator compound(as determined by conducting the frontal chromatography of the indicatorcompounds in the absence of the compound library) indicates that thefunctional target receptor is being inhibited by one or more members ofthe compound library. When a compound library is identified as having aninhibitor present in the library for the functional target receptor, thelibrary can be further analyzed using FC-MS to identify the ligandsbinding to the target receptor.

[0156] When using an indicator compound is employed, the break throughtime for the indicator compound is first determined by applying theindicator compound and the void marker compound to the column containingthe target receptor under frontal chromatography conditions. The columnis then typically equilibrated or partially equilibrated with thecompound library to be screened. Generally, the compound library isapplied or infused into the column for a time sufficient to allow all ofthe library members to break through the column. The effluent duringthis period may be presented to the mass spectrometer for analysis ormay be collected for recycling or disposal. Once the column has beenequilibrated or partially equilibrated with the compound library, amixture comprising the compound library, void marker compound and theindicator compound is applied to or infused into the column using thefrontal chromatography procedures described herein. Preferably, theindicator compound will be present in the mixture in a concentrationless than its K_(d) value. Typically, the indicator compound will bepresent in an amount ranging from about 1 nM to about 10 μM, morepreferably from about 10 nM to about 1 μM. The effluent from the columnis analyzed to determine the break through time for the indicatorcompound in the presence of the compound library and this time period iscompared to the pre-determined break through time for the indicatorcompound to ascertain whether the compound library has an affinity forthe target receptor.

[0157] Alternatively, the indicator compound and the void markercompound, without the compound library, can be applied or infused intothe column after equilibration or partial equilibration of the columnwith the compound library. This technique allows very strongly boundligands or those with slow off rates to be detected.

[0158] An indicator compound can also be useful in determining whetherthe overall affinity of a compound library is due to the presence of aplurality of weak binders or to one or more strong binders. In thisembodiment, a column equilibration procedure is initiated by infusion ofthe compound library as described herein. The indicator compound, in thepresence of the library and the necessary void markers, is then passedthrough the column during the initial stages of the equilibrium process(typically at about 1 to about 5 minutes) and a first break through timefor the indicator compound is determined. The flow of compound librarywithout the indicator is then reestablished through the column. After aperiod of time, the indicator/library/void marker solution is againpassed through the column and a second break through time is determined.When the same indicator compound is used through this procedure, theintervening time period between each application of the indicatorcompound can be as short as the time necessary to wash off the indicatorcompound, and as long as the total equilibration time (e.g., 1 min to 60minutes, respectively). This cycle can be repeated any number of times.In this method, the discrimination between weak and strong bindersoccurs because a weak ligand will reach equilibrium on the column soonerthan a strong one. Monitoring of the column activity during theequilibration process is illustrated in FIG. 11 which is a graph of thereduction of column activity as a function of time for two differentlibraries: one containing many weak binders and one containing strongbinders. Even though the same overall reduction in column activity isachieved (as measured by the indicator compound), the rate of reductionis slower for the strong ligands compared to the weak ligands.

[0159] An alternative method for distinguishing between a plurality ofweak binders and one or more strong binders in a compound library isillustrated in FIG. 10. In this embodiment, an indicator compound(V_(n-1) in FIG. 10) is first selected having an affinity for the targetreceptor which is weaker than the putative ligands of interest (V_(n))but stronger than those not of interest (V_(1,max)). A mixturecomprising the compound library, the void marker compound and theindicator compound at a pre-determined initial concentration and signalintensity is then applied or infused into a column comprising the targetreceptor under frontal chromatography conditions to provide an effluent.In this embodiment, the column is not pre-equilibrated with the compoundlibrary. Preferably, the concentration of the indicator compound in themixture is greater than or equal to its dissociation constant for thetarget receptor. The effluent from the column is then analyzed by massspectrometry to determine a break through time for the indicatorcompound and its signal intensity. In the presence of a plurality ofweak binders (i.e., weaker than the indicator compound), the breakthrough time for the indicator will be less than its break through timein the absence of the compound library and the basic shape of the curvewill be unchanged. In the presence of one or more ligands having anaffinity for the target receptor greater than the indicator compound,the break through time will also be less than the break through time forthe indicator compound in the absence of the compound library, but theshape of the curve will also display a “roll up” effect as illustratedin FIG. 10. This “roll up” effect is due to the removal of boundindicator compound by the stronger ligand(s). Thus, for a short periodof time, the concentration of the indicator compound is higher than itsinfusion concentration until the stronger ligand(s) breaks through. Theamount of bound indicator compound removed is dependent upon the K_(d)value and the concentration of the stronger ligand(s) present in thelibrary. Weaker ligands do not exert this “roll up” effect because theypropagate through the column more quickly than the indicator compound.Detection or measurement of this “roll up” or bump in the break throughcurve (typically measured as a change in signal intensity) indicates thepresence of ligands in the compound library having an affinity for thetarget receptor greater than the indicator compound. The detection ormeasurement of a “roll up” effect may also be used when screening targetreceptors for affinity to an immobilized ligand(s).

[0160] In addition to detecting the indicator compound using massspectrometry, other methods of detection may also be employed. Anydetection method that can measure the indicator compound over thebackground signal of the library compounds can be used. For example, anindicator compound can be detected in the effluent from the columnusing, by way of example, fluorescence, infra-red absorption, UV-visibleabsorption, nuclear magnetic resonance (NMR), atomic spectroscopy (i.e.,atomic adsorption spectroscopy (AAS), inductively coupled plasma-opticalemission spectroscopy (ICP-OES), etc.), flow cytometry, electrochemicaldetection and the like. Procedures and apparatus for detecting compoundsusing such methods are well-known in the art and any conventionalprocedures and apparatus may be used.

[0161] The apparatus of this invention allow a plurality of FC-MSanalyses to be conducted simultaneously using a single mass spectrometerto intermittently monitor each column. Unlike “capture and release”methods which typically provide an elution peak or “spike” for eachligand, FC-MS does not require constant effluent monitoring because oncea library member breaks through the column, that member is continuouslypresent in the effluent and can be detected by the mass spectrometer.Therefore, a plurality of FC-MS analyses can be conducted simultaneouslyusing a single mass spectrometer to intermittently monitor each column.For example, using this invention, at least about 100 columns can beconducted simultaneously.

[0162] When employing multiple columns, each column is typicallymonitored for a brief period of time before switching to the nextcolumn. For example, with a quadrupole mass spectrometer, each column istypically monitored sequentially for a period of about 0.5 seconds toabout 10 seconds, preferably for about 1 second to about 5 seconds,before switching to the next column. The effluent from each column isanalyzed as described herein using mass spectrometry. Generally, asingle data file is used to collect all of the data from the multiplecolumn thereby generating a composite total ion chromatogram.Subsequently, separate total ion chromatograms for each column arerecreated by synchronizing column switching with mass spectrometry dataacquisition.

[0163] In a preferred embodiment, each column will have an individualelectrospray needle for injection of the column's effluent into theelectrospray mass spectrometer. Any geometric arrangement of multipleelectrospray needles that allows for fast and repetitive sequences ofneedle advancement may be employed. A suitable apparatus for theinjection of multiple effluents into an electrospray mass spectrometeris described in U.S. patent application No. 09/069,656, filed Apr. 29,1998, and U.S. Pat. No. ______, filed on even date herewith, as AttorneyDocket No. 026579-260 and entitled “Device for Delivery of MultipleLiquid Sample Streams to a Mass Spectrometer,” the disclosures of whichare incorporated herein in their entirety. Alternatively, a linearmoving row of electrospray needles (sprayers) and the like may beemployed. See, for example, Q. Xue et al., Anal. Chem. 1997, 69, 426-430and references cited therein, the disclosed of which is incorporatedherein by reference in its entirety.

[0164] A representative apparatus for screening compound libraries usinga plurality of columns is illustrated in FIG. 2. As shown in FIG. 2,each of a plurality of columns 13 is connected via tubing 14 and tee 15to a first reservoir 16, containing a solution of a compound library ina binding buffer, and a second reservoir 17, containing the bindingbuffer. In FIG. 2, reservoirs 16 and 17 are syringes although anysimilar reservoir may be employed. Each column 13 contains a targetreceptor bound to a solid phase support. The buffer solution inreservoir 17 is used to wash column 13 before or after introduction ofthe compound library. The outflow end of each column 13 is connected toa mixing tee 18, which is also connected to reservoir 19, containing asupplemental diluent, via tubing 20. The effluent from each column 13 ismixed with the supplemental diluent from reservoir 19 in mixing tees 18and the outflow is directed via tubing 20 and valves 21 into anelectrospray mass spectrometer 22, via an electronically-actuatedmulti-port selection valve 23, or into waste/recovery containers 24. Tocontrol the flow from reservoirs 16, 17 and 19, pressure is applied toplungers 25 via, for example, pumps.

[0165] Alternatively, in another embodiment illustrated in FIG. 3, theoutflow from mixing tees 18 may be directed via tubing 20 intoindividual electrospray needles 26 for mass spectrometer analysis.

[0166] When using an indicator compound, sequential runs of multiplecolumns may be advantageous since this allows the retention time for theindicator compound to be more accurately determined. Parallel infusionof the indicator through a plurality of columns is feasible provided anapparatus is used with a suitable high sampling rate (e.g., allowing fora minimum of five mass spectral measurements on the break through curveof the indicator for each of the columns. Such an apparatus is describedin U.S. patent application No. 09/069,656, filed Apr. 29, 1998, and U.S.Pat. No. ______, filed on even date herewith, as Attorney Docket No.026579-260.

[0167] A representative apparatus for sequentially screening compoundlibraries with a indicator compound using a plurality of columns isillustrated in FIGS. 2, 3 and 4. The apparatus shown in FIGS. 2 and 3are preferred and are employed as described herein for FC/MS. However,reservoir 16 optionally contains a solution of the compound library plusthe indicator compound and void marker compounds in a binding buffer,while reservoir 17 optionally contains a solution of only the compoundlibrary in the binding buffer. Alternatively, the apparatus illustratedin FIG. 4 can be used. As shown in FIG. 4, a plurality of reservoirs 27(e.g., syringes) are held in place with clamp 38. Each reservoir 27contains a mixture of a compound library and an indicator compound in asuitable diluent (or, alternatively, simply the indicator). The end ofeach reservoir 27 is connected via tubing 29 to the inflow end of acolumn 30 containing the target receptor bound to a solid phase support.The outflow end of each column 30 is connected via tubing 31 to anelectronically-actuated multiport stream selection valve 32 whichcontrols the flow of the effluent from columns 30. Using valve 32, theeffluent from the columns may be directed into a waste container 33, viatubing 34, or into mixing tee 35, via tubing 36. Mixing tee 35 is alsoconnected to reservoir 36, containing a supplemental diluent, via tubing37. The effluent from each column 30 is mixed with the supplementaldiluent from reservoir 36 in mixing tee 35 and the outflow is directedvia tubing 38 into an electrospray mass spectrometer 39. To control theflow from the reservoirs 27 into columns 30, a stand-off block 40 may beemployed. When pressure is applied to stand-off block 40 via, forexample, a pump, the plunger 41 of each reservoir 27 is individuallydepressed in sequence thereby infusing the contents of the reservoirthrough tubing 29 into the corresponding column 30. The effluentemerging from each column 30 is sequentially directed into massspectrometer 39 for analysis.

[0168] The apparatus of this invention also permit the absolute affinityor dissociation constant, K_(d), for certain individual members of acompound library to be readily determined. In this regard, ligandshaving an affinity for the target receptor break through the column atvolumes (i.e., break through times) related to their concentrations andK_(d) values, according to the following equation:${V_{x} - V_{0}} = \frac{B_{t}}{\lbrack X\rbrack_{0} + \left( K_{d} \right)_{x}}$

[0169] where B_(t) represents the dynamic binding capacity of thecolumn; [X]₀ is the infusion concentration of the ligand in the compoundlibrary; K_(d) is the dissociation constant for the ligand; V₀ is thevoid volume; and V_(x) represents the volume at the mid-point of thefront corresponding to the break through of the ligand. This simpleequation indicates that, once B_(t) and the concentration of the ligandare known, the dissociation constant of a ligand can be determined froma single measurement of its V_(x)-V₀. This equation strictly appliesonly in the case of a single ligand. In many cases, however, thisequation or a modification of it can be applied to multiple ligands aswell.

[0170] In order to determine B_(t), a representative compound, e.g.,compound X, is infused through the column at various concentrations andthe corresponding V_(x)-V₀ values measured. A plot of ([X](V-V₀))⁻¹versus [X]⁻¹ is generated, where the y-intercept indicates the dynamicbinding capacity of the column (B_(t)) (analogous to a Lineweaver-Burkplot).

[0171] Once the dynamic binding capacity of the column has beendetermined, the dissociation constants for individual members of thecompound library can be determined from a single FC-MS run. For example,the K_(d) for compounds where [X]<<(K_(d))_(x) is determined simply fromB_(t)/(V-V₀). For those members of the library with a low dissociationconstant, knowledge of their concentration or infusion of the compoundlibrary at higher dilution is required to determine K_(d).

[0172] The following examples are offered to illustrate this inventionand are not to be construed in any way as limiting the scope of thisinvention. Unless otherwise stated, all temperatures are in degreesCelsius.

EXAMPLES

[0173] In the examples below, the following abbreviations have thefollowing meanings. If an abbreviation is not defined, it has itsgenerally accepted meaning.

[0174] B_(t)=dynamic binding capacity

[0175] °C.=degrees Celsius

[0176] cm=centimeter

[0177] eq.=equivalents

[0178] FAB=fast atom bombardment

[0179] FC=frontal chromatography

[0180] g=grams

[0181] K_(d)=dissociation constant

[0182] L=liter

[0183] MALDI=matrix-assisted laser desorption/ionization

[0184] meq.=milliequivalent

[0185] mg=milligram

[0186] mL=milliliter

[0187] mM=millimolar

[0188] mmol=millimole

[0189] MS=mass spectrometry

[0190] m/z=mass charge ratio

[0191] N=normal

[0192] PBS=phosphate buffered saline

[0193] PEEK=poly(ether ether ketone)

[0194] pmol=picomole

[0195] TIC=total ion chromatogram

[0196] μg=micrograms

[0197] μL=microliter

[0198] μm=micrometer

[0199] μM=micromolar

[0200] V₀=void volume

Example 1 Screening of an Oligosaccharide Library Using FC-MS

[0201] In this example, a compound library containing a mixture of sixoligosaccharides was screened using frontal chromatography incombination with an electrospray mass spectrometer to determine therelative affinity of the oligosaccharides for a monoclonal antibody thatrecognizes the 3,6-dideoxy-D-galactose (abequose) epitope in Salmonellaparatyphi B O-antigens.

[0202] The compound library consisted of the following sixoligosaccharides: αGalNAc(1→3)βGal-OGr (compound 1);αGal(1→3)[αFuc(1→2)]βGal-OGr (compound 2); αMan(1→3)[αMan(1→6)]PMan-OGr(compound 3); αAbe(1→3)αTal-OCH₃ (compound 4);αGal(1→2)[αAbe(1→3)]αMan-OCH₃ (compound 5); andαGlc(1→4)βGlc(1→4)αGal(1→2)-[αAbe(1→3)]αMan(1→3)αGlc(1→4)βGlc-OCH₃(compound 6), wherein Gr=O(CH₂)₈CO₂CH₃. Compound 1-3 were obtained usingthe procedures described in U.S. Pat. No. 4,362,720 to R. U. Lemieux etal., issued Dec. 7, 1987; U.S. Pat. No. 4,137,401 to R. U. Lemieux etal, issued Jan. 30, 1979; and K. J. Kaur et al., “Use ofN-Acetylglucosaminyltransferases I and II in the Preparative Synthesisof Oligosaccharides”, Carbohydr. Res. 1991, 210, 145-153; respectively,the disclosures of which are incorporated herein by reference in theirentirety. Compounds 4-6 were obtained using the procedures described inD. R. Bundle et al., “Modulation of Antibody Affinity by SyntheticModifications of the Most Exposed Pyranose Residue of A TrisaccharideEpitope”, Bioorg. Med. Chem. 1994, 2, 1221-1229, the disclosure of whichis incorporated herein by reference in its entirety. Compounds 1-3 areknown to have no specificity for the antibody. On the other hand,compounds 4-6 contain the minimal requirement for recognition (abequose)and span a range of affinity for the antibody. The K_(d) values forcompounds 4-6, as determined by titration microcalorimetry, are shown inTable 1 below.

[0203] The monoclonal antibody used in this experiment was produced asdescribed in D. R. Bundle et al, “Molecular Recognition of a SalmonellaTrisaccharide Epitope by Monoclonal Antibody Se155.4” Biochem. 1994, 33,5172-5182. The antibody (0.5 mg) was biotinylated with a biotin reagentcontaining a long-chain spacer arm (NHS-LC-biotin, Pierce). The extentof biotin incorporation was monitored by matrix-assisted laserdesorption/ionization and the reaction was terminated at 14 biotins/IgG(average). The biotinylated antibody was then coupled to a beadedsupport by incubating the antibody with 25 μL of Ultralink immobilizedavidin (Pierce, Cat. No. 53119) in bicarbonate buffer (pH 8.5) for 1hour. The beads were then thoroughly washed with the bicarbonate buffer.A UV quantitation indicated an immobilization of ˜45 μg antibody/25 μLbeads was achieved. The beads were then slurry-packed into a 500 μm i.d.by 11.5 cm poly(ether ether ketone) (PEEK) column body (˜23 μL columnvolume).

[0204] In this experiment, a mixing tee served a dual role as a columnend-fitting and mixing chamber for the column eluent and organic make-upflow. The column was then directly connected to an electrospray massspectrometer (Hewlett-Packard series 1100 MSD, single quadrupole).

[0205] For operation in frontal chromatography mode, the column wasfirst flushed with ammonium acetate buffer (NH₄OAc, 2 mM, pH 6.7). Afterflushing , the flow was switched to a second solution containing amixture of the six oligosaccharides in ammonium acetate buffer, eachpresent at 1 μM. All solutions were infused concurrently with amulti-syringe pump (PHD 200, Harvard Apparatus) at a flow rate of 8μL/min/syringe (1 cc syringes). A Rheodyne valve (Model 9725) was usedfor flow switching. The column effluent combined with the make-up flow(10% 2 mM NH₄OAc buffer in acetonitrile) in the tee to provide a flowrate of 16 μL/min into the mass spectrometer.

[0206] For the analysis of this mixture, the spectrometer was scannedfrom m/z 100-1500. Data was collected in scan mode with positive iondetection. A total ion chromatogram (TIC) was constructed from a 50minute run time as shown in FIG. 5A. This represented the consumption ofonly 400 pmol of each oligosaccharide. Peaks at specific m/z values werethen identified through the analysis of the mass spectra giving rise tothe TIC and selected ion chromatograms for all six compounds werereconstructed from the TIC as shown in FIG. 5B. Compounds 1-3 breakthrough the column simultaneously as indicated by the solid line. Massspectra were then generated from time-slices of the TIC (at times I, IIand III) as shown in FIGS. 5C, 5D and 5E. These mass spectra chart theprogression of the various oligosaccharides through the column. Aspectrum representing the onset of compound 4 is not shown.

[0207] As discussed above, ligands having no affinity for the targetreceptor break through at the void volume (V₀), while compounds havingan affinity for the target ligand break through later, at volumesrelating to their concentrations and K_(d) values, according to thefollowing equation:${V_{x} - V_{0}} = \frac{B_{t}}{\lbrack X\rbrack_{0} + \left( K_{d} \right)_{x}}$

[0208] where B_(t) represents the dynamic binding capacity of thecolumn; [X]₀ is the infusion concentration of the ligand in the compoundlibrary; K_(d) is the dissociation constant for the ligand; V₀ is thevoid volume; and V_(x) represents the volume at the mid-point of thefront corresponding to the break through of the ligand.

[0209] In order to determine B_(t), compound 5 was infused through thecolumn at various concentrations and the corresponding V-V₀ valuesmeasured. A plot of ([A]₀(V-V₀))⁻¹versus [A]₀ ⁻¹ was generated, where Ais compound 5, as shown in FIG. 6. The y-intercept indicated a B_(t) of520 pmol. Each antibody molecule contains two binding sites, thereforethis corresponds to an active capacity of 260 pmol of protein(representing 93% of the total amount of protein bound). The x-interceptindicated a K_(d) of 11.2 μM for compound 5, which compares favorablywith the value determined by microcalorimetry as shown in Table 1.

[0210] Knowledge of the column capacity prior to the screening of amixture allows for the determination of dissociation constants from asingle frontal chromatogram. For compounds with [X]<<(K_(d))_(x), theK_(d) can be determined simply from B_(t)/(V-V₀). For example, compound4 was shown to have a K_(d) of 0.2 mM, as determined from thechromatogram of FIG. 5B. Compounds with low dissociation constantsrequire either the knowledge of their concentration or the infusion ofthe mixture at higher dilution for the determination of K_(d). The K_(d)of compound 6, at a 1 μM concentration, was determined from the samechromatogram to be 1.5 μM.

[0211] The column was regenerated offline by washing with a large volumeof binding buffer. The column used in this example was subjected to over150 runs with no observable loss of activity or leaching of theantibody.

[0212] The results from this experiment are shown in Table 1. TABLE 1K_(d) ± s (μM)² Oligosaccharide Micro- FC/MS No. Gr = O(CH₂)₈CO₂CH₃(MNa)⁺¹ cal³ Ind.⁴ Mix⁵ 1 αGalNAc(1→3)βGal—OGr 576.3 — — 2αGal(1→3)[αFuc(1→2)]βGalO—Gr 681.3 — — 3 αMan(1→3)[αMan(1→6)]βMan—OGr697.3 — — 4 αAbe(1→3)αTal—OCH₃ 347.0 190 185 ± 17  178 ± 23  5αGal(1→2)[αAbe(1→3)[αMan—OCH₃ 509.2 6.3 12.6 ± 1.3  10.2 ± 1.1  6αGlc(1→4)βGlc(1→4)αGal(1→2)[αAbe 1157.4 0.88 1.79 ± 0.20 1.71 ± 0.16(1→3)]αMan(1→3)αGlc(1→4)βGlc- OCH₃

[0213] The results in Table 1 demonstrate that the affinity of variousputative ligands in a compound library for a target receptor can bedetermined relative to other putative ligands in the library; and thatthe dissociation constant, K_(d), for putative ligands and the targetreceptor can be determined. The results further demonstrate that thereis an acceptable correlation between the literature K_(d) values andthose generated by FC-MS procedures.

Example 2 Screening of an Oligosaccharide Library Using FC-MS and anIndicator Compound

[0214] In this example, the use of an indicator compound to screen acompound library is demonstrated. The antibody used in this example wasthe same as that used in Example 1, i.e., a monoclonal antibody thatrecognizes the 3,6-dideoxy-D-galactose (abequose) epitope in Salmonellaparatyphi B O-antigens. The column was also essentially the same as thecolumn in Example 1 and it was prepared and operated as describedtherein.

[0215] In this experiment, three solutions were prepared. Solution Acontained the following four oligosaccharide in 2 mM NH₄OAc:αGalNAc(1→3)βGal-OGr (compound 1); αGal(1→3)[αFuc(1→2)]βGal-OGr(compound 2); αMan(1→3)[αMan(1→6)]βMan-OGr (compound 3);αAbe(1→3)αTal-OCH₃ (compound 4), wherein Gr=O(CH₂)₈CO₂CH₃. Solution Bcontained αGal(1→2)[αAbe(1→3)]αMan-OCH₃ (compound 5) in 2 mM NH₄OAc; andSolution C contained compounds 1-5 in 2 mM NH₄OAc. In all solutions,compounds 1, 2 and 3 were present at 1 μM, compound 4 was present at0.16 μM, and compound 5 was present at 15 μM. In this example, compound4 was used as the indicator compound and compound 5 was used torepresented a member of a compound library. The remaining compounds wereused to determine V₀.

[0216] Solution A containing compounds 1-4 was infused into the columnas described in Example 1. A quadrupole mass spectrometer was used tomonitor the effluent. The mass spectrometer was operated in selected ionmonitoring (SIM) mode, on the (M+Na)⁺peak of each compound. FIG. 5Ashows the selected ion chromatograms generated from an infusion ofcompounds 1-4 (i.e., Solution A). The breakthrough volume for compound 4was 3.0±0.1 μL. The column was regenerated by flushing with the bindingbuffer (i.e., 2 mM NH₄OAc) for about 10 min. at which time essentiallyall traces of compound 4 were removed.

[0217] Using the apparatus of FIG. 1, Solution B (compound 5) andSolution C (compounds 1-5) were loaded into separate syringes. SolutionB was infused through the column until dynamic equilibrium for compound5 was attained. At this point, the flow was switched to the syringecarrying Solution C, and the selected ion chromatograms of FIG. 7B weregenerated using the quadrupole mass spectrometer. As shown in FIG. 7B,pre-equilibration of the column with compound 5 leads to a measurableshift in the breakthrough volume of the indicator compound 4 (to 1.1±0.3μl). This is consistent with the fact that compound 5 is a ligand havinga K_(d) for the antibody lower than that of the indicator compound 4(see Table 1 above). Therefore, by simply monitoring the indicatorcompound, the fact that the representative library contained a compoundwith a higher affinity for the target receptor was readily apparent.

[0218] Note that while the indicator compound (compound 4) in thisexperiment was added to a solution of the representative library(compound 5), this will not always be necessary. In those situationswhere the library (Solution B) contains a strongly retained compound(i.e., low K_(d), or off-rate), Solution A can be substituted forSolution C (i.e., the indicator does not need to be mixed with thelibrary).

Example 3 Screening of an Oligosaccharide Library Using FC-MS

[0219] In this example, a compound library containing a mixture of fouroligosaccharides was screened using frontal chromatography incombination with an electrospray mass spectrometer to determine therelative affinity of the oligosaccharides for cholera toxin B subunit.

[0220] The compound library consisted of the following fouroligosaccharides: αGalNAc(1→3)βGal-OGr (compound 1);αGal(1→3)[αFuc(1→2)]βGal-OGr (compound 2); αMan(1→3)[αMan(1→6)]βMan-OGr(compound 3); and GM₁ oligosaccharide (compound 7, whereinGr=O(CH₂)₈CO₂CH₃. Compound 7, which is the natural ligand for choleratoxin B subunit, was obtained using the procedures described in A. Schönet al., “Thermodynamics of Intersubunit Interactions in Cholera Toxinupon Binding to the Oligosaccharide Portion of Its Cell SurfaceReceptor, Ganglioside G_(M1)” Biochem. 1989, 28, 5019-5024, thedisclosure of which is incorporated herein by reference in its entirety.Cholera toxin B subunit was obtained from LIST Biochemicals, Campbell,Calif.

[0221] A column was prepared from a 12 cm section of 0.01″ (250 μm) i.d.PEEK tubing (column volume of about 6 μL). The column was packed withPOROS 20 immobilized streptavidin particles (available from PerseptiveBiosystems, Framingham, Mass.).

[0222] Cholera toxin B subunit (a pentameric protein) was biotinylatedto provide about 1-2 biotins/monomer, as measured by MALDI. A dilutesolution of this biotinylated protein (4 μM) was infused through thepre-packed column such that the total amount of cholera toxin B subunitbound was approximately 200 pmol after washing (as determined by UVquantitation).

[0223] A solution containing compounds 1-3 and 7 was prepared. Allcompounds were present at 2 μM, in 2 mM NH₄OAc (pH 6.9). Using anapparatus similar to that shown in FIG. 1, the column was firstequilibrated with the binding buffer (2 mM NH₄OAc). The solutioncontaining compounds 1-3 and 7 was then infused through the column at 8μL/min. The effluent was combined with a typical make-up flow (10% 2 mMNH₄OAc in acetonitrile) and passed into an electrospray singlequadrupole mass spectrometer. Data was collected in scan mode, withnegative ion detection.

[0224] A total ion chromatogram was generated, followed byreconstruction of selected ion chromatograms for each of compounds 1-3and 7 as shown in FIG. 8. As illustrated in FIG. 8, compounds 1-3 brokethrough in the void volume of the system (˜4 min×8 μL/min=32 μL) whilecompound 7 (GM₁ oligosaccharide) broke through at ˜300 μL. Thus, GM₁oligosaccharide (K_(d)≅100 nM) has a stronger affinity for cholera toxinB subunit than compounds 1-3 which have little or no affinity forcholera toxin B subunit.

[0225] A second mixture was then prepared in the binding buffer andanalyzed by FC-MS in a similar fashion. This mixture contained asynthetically prepared GM₁ analogue, i.e.,βGal(1→3)βGalNAc(1→)-OCH₂CH₂O-(←2)αNeu5Ac, (compound 8) in an impureform (i.e. containing unidentified intermediates and reactionbyproducts). Compound 8 was prepared by the methods described in P.Fügedi et al, “A Novel Promoter for the Efficient Construction of1,2-trans Linkages in Glycoside Synthesis, Using Thioglycosides asGlycosyl Donors” Carbohydr. Res. 1986, 149, C9-C12; A. Marra et al.,Stereoselective Synthesis of 2-Thioglycosides of N-AcetylneuraminicAcid”, Carbohydr. Res. 1989, 187, 35-42; and L. Lay et al., “Synthesisof the Propyl Glycoside of the Trisaccharideα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GalpNAc. Components of a Tumor AntigenRecognized by the Antibody Mbr1” Helv. Chim. Acta. 1994, 77, 509-514;the disclosures of which are incorporated herein by reference in theirentirety. The mixture was infused through the column, and the massspectrometer was set to operate in selected ion monitoring mode, onnegative ions representative of compounds 3 and 8. Selected ionchromatograms were generated for these ions as shown in FIG. 9. FIG. 9shows that compound 3 broke through in the void volume (m/z 673.2). Amore complex pattern was observed for the ions with a mass/charge of717.2 u. A certain fraction of these ions also broke through in the voidvolume (˜25%), while the remaining 75% broke through significantly later(at about 11 min). This two-front profile indicates an isobaric impurityexists at the 25% level, which does not bind to cholera toxin B subunit.Thus, FC-MS is able to ascertain the presence of isobaric, non-bindingimpurities. Reasonably accurate quantitation of these impurities canalso be achieved.

Example 4 Screening of a Compound Library Containing 100 PutativeLigands Against a Human Enzyme

[0226] In this example, a compound library containing a mixture of 100tripeptides was screened against immobilized human α-thrombin usingfrontal chromatography in combination with an electrospray massspectrometer. The peptides were synthesized as a mixture by establishedsolid phase techniques and were purchased from Alberta Peptide Institute(Edmonton, Alberta, Canada). This set of peptides all have a commonC-terminal amino acid (arginine), while the remaining two positions arerandom and chosen from a set of 10 amino acids (see FIG. 12). FIG. 12also displays an electrospray mass spectrum of the mixture. The spectrumwas collected from an infusion of a 50 μM solution in 1:1acetonitrile:ammonium acetate (2 mM, pH 7.2). Assuming equimolar ratiosof all peptides, this corresponds to 0.5 μM per peptide. This spectrumhighlights a peak at m/z of 419.2. This peak corresponds to two isomericentries in the library: PfR and fPR, where f refers to D-phenylalanine.The tripeptide fPR has been identified in the literature as an inhibitorpossessing a K_(d) value of approximately 1 μM against human α-thrombin.An experiment was conducted to determine if this ligand could bedetected when present in the full mixture as screened against a thrombincolumn.

[0227] A column was prepared from a 5 cm section of 0.01″ (250 μm i.d.)PEEK tubing (column volume of about 2.5 μL). The column was packed withPOROS 20 immobilized streptavidin particles (available from PerseptiveBiosystems, Framinghan, Mass.). Human α-thrombin was purchased fromSigma Chemical Co., and biotinylated with a reagent containing along-chain spacer arm (sulfo-NHS-LC-biotin, Pierce). The extent ofbiotin incorporation was less than 5 biotins/thrombin, as monitored bymatrix-assisted laser desorption/ionization. A dilute solution of thisbiotinylated protein (approximately 2 μM) was infused through thepre-packed column in the presence of 0.1% bovine serum albumin (w/v)such that the total amount of immobilized thrombin was approximately 570pmol (as calculated by a determination of the reduced capacity of thecolumn for free biotin).

[0228] The BOC-protected fPR (BOC-fPR, also known as ligand) waspurchased from Calbiochem and infused through the column at aconcentration of 1 μM in ammonium acetate solution (2 mM, pH 7.2), andat a flow rate of 8 μL/min. FIG. 13 displays a chromatogram reflectingthree infusions/wash cycles of this peptide through the column, in thepresence of a void marker compound (a non-binding trimannosyl compound).FIG. 13 shows the selected ion chromatograms for each compound. Theaverage V-V₀ value was determined to be 4.66 μL. Based on thisexperiment, and the infusion of this peptide at higher concentrations,the B_(t) value of the column was calculated to be approximately 145pmol (˜25% active). The BOC-fPR was selected as the indicator compoundfor the following work, with the trimannosyl compound the correspondingvoid marker compound.

[0229] The library of peptides was then infused through the column at aconcentration of 1 μM per peptide for approximately 30 minutes,whereupon the indicator compound and void market compound (in thepresence of the library) were infused. The same buffer and flow rateconditions as above were used. FIG. 14 displays the V-V₀ valueimmediately before (14A) and immediately after (14B) the 30 minuteequilibration time. The drop in V-V₀ as a result of the 100 peptidesindicates the loss of virtually all of the binding activity of theprotein.

[0230] To determine the nature of the compounds giving rise to theindicator shift, the peptide library was infused through a fresh,identically prepared column in FC/MS mode. The HP quadrupoleelectrospray mass spectrometer was set to scan the mass range from m/z100 to 600 at a scan rate of approximately 1 sec/cycle. The effluent wasmonitored in real time, and the experiment was stopped at approximately15 minutes. The results are displayed in FIGS. 15A and 15B. FIG. 15Ashows a featureless total ion chromatogram. However, a generation ofselected ion chromatograms from the peaks representing the mixturecomponents resulted in the identification of m/z 419.2 as giving rise tothe largest V-V₀ shift (as shown in FIG. 15B). Two breakthrough curvesare evident in this Figure, indicating the presence of at least twoisomers. Based on a knowledge of the mixture composition, this isconsistent with the presence of the isomers PfR (non-binding) and fPR(the ligand). A fresh column was constructed through which a solution ofjust the void marker compound and fPR (1 μM each) was infused. Thisgenerated a V-V₀ that was approximately twice the value measured fromthe mixture. A similar experiment was conducted using PfR instead, whichgenerated no measurable V-V₀. This confirms that fPR is indeed theligand in the mixture. The combination of a large indicator shift and asmaller than expected V-V₀ for the ligand is consistent with thepresence of additional ligands. Thrombin is a serine protease capable ofcleaving the C-terminal side of K and R, therefore a large fraction ofthese peptides serve as substrates for the enzyme. At the infusionconcentrations of the experiment, these peptides compete with thebinding of fPR. This shows that FC/MS, in conjunction with the indicatoranalysis, serve as a rapid means of identifying ligands from mixtures.

[0231] An additional experiment was conducted with this system toillustrate the use of the roll-up effect in determining the presence ofa strong ligand. A thrombin column similar in construct to thosementioned above was prepared, this time containing approximately 50 pmolof active protein. The ligand fPR was selected as an indicator andinfused through the column at a concentration of 1 μM to generate theselected ion chromatogram of FIG. 16A. This represents a typical breakthrough curve. The column was regenerated offline with binding buffer,whereupon a solution containing 1 μM of fPR and 1 μM of fPR-chloromethylketone (an affinity label with a K_(d) value of approximately 50 nM) wasinfused. The selected ion chromatogram is shown in FIG. 16B, where thesolid line represents fPR and the dashed line fPR-chloromethyl ketone.For clarity, the selected ion chromatogram for the void marker is notdisplayed. Firstly, there is a time shift in the break through curve forfPR vs. the break through curve of FIG. 16A. Secondly, the y axis ofFIG. 16B indicates a maximum intensity of approximately 3 times that ofthe infusion concentration, followed by a return to the infusionconcentration level. This indicates the presence of a stronger ligand inthe mixture that causes the release of prebound indicator (thisindicator loaded on to the column ahead of the stronger ligand). Notethat the peak correlates with the onset of the break through time forthe stronger ligand (dashed line). Therefore, monitoring solely theindicator leads to the identification of a mixture containing at leaston ligand with a binding constant lower than the indicator.

[0232] From the foregoing description, various modifications and changesin the composition and method will occur to those skilled in the art.All such modifications coming within the scope of the appended claimsare intended to be included therein.

What is claimed is:
 1. An apparatus for screening a compound library todetermine the relative or absolute affinity of a plurality of putativeligands to a target receptor or a plurality of target receptors, whichapparatus comprises: (a) a column comprising a target receptor or aplurality of target receptors, each target receptor optionally attachedto a solid phase support, and having a inflow end and an outflow end,wherein said column is capable of having a compound library comprising aplurality of putative ligands applied thereto under frontalchromatography conditions to produce an effluent from the outflow end ofthe column; (b) a first reservoir connected to the inflow end of saidcolumn for applying the compound library to the column; (c) a massspectrometer connected to the outflow end of said column forcontinuously or intermittently analyzing the effluent from the column.2. The apparatus of claim 1 , wherein said apparatus further comprises:(d) a second reservoir connected to the inflow end of the column forapplying either (i) a mixture comprising the compound library, at leastone void marker compound and an indicator compound or a plurality ofindicators compounds, (ii) at least one void marker compound and anindicator compound or a plurality of indicator compounds, or (iii) abuffer solution to the column.
 3. The apparatus of claim 1 , whereinsaid apparatus further comprises: (e) a third reservoir connected to theoutflow end of the column for supplying a supplemental diluent to theeffluent before analysis by the mass spectrometer.
 4. The apparatus ofclaim 1 , wherein the column has an internal diameter ranging from about10 μm to about 4.6 mm.
 5. The apparatus of claim 4 , wherein the columnhas an internal diameter of from about 100 μm to about 250 μm.
 6. Theapparatus of claim 1 , wherein the column has a length of from about 1cm to about 30 cm.
 7. The apparatus of claim 1 , wherein the column hasa length of from about 2 cm to about 20 cm.
 8. The apparatus of claim 1, wherein each target receptor is independently selected from the groupconsisting of proteins, glycoproteins, glycosaminoglycans,proteoglycans, integrins, enzymes, lectins, selecting, cell-adhesionmolecules, toxins, bacterial pili, transport proteins, receptorsinvolved in signal transduction or hormone-binding, hormones,antibodies, major histocompatability complexes, immunoglobulinsuperfamilies, cadherins, DNA or DNA fragments, RNA and RNA fragments,whole cells, cell fragments, tissues, bacteria, fungi, viruses,parasites, preons, and synthetic analogs or derivatives thereof.
 9. Theapparatus of claim 1 , wherein the target receptor is bound to a solidphase support.
 10. The apparatus of claim 9 , wherein the targetreceptor is covalently bound to the solid phase support or bound viabiotin-avidin or biotin-streptavidin binding.
 11. The apparatus of claim9 , wherein the solid phase support is selected from the groupconsisting of polymeric beads, polymeric gels, glass beads, silicachips, silica capillaries, agarose, diatomaceous earths and pulp. 12.The apparatus of claim 1 , wherein the column contains from about 1 fmolto about 10 nmol of target receptor active sites.
 13. The apparatus ofclaim 1 , wherein the mass spectrometer is an electrospray massspectrometer.
 14. An apparatus for screening a plurality of compoundlibraries to determine the relative or absolute affinity of a pluralityof putative ligands in each library to a target receptor or a pluralityof target receptors, which apparatus comprises: (a) a plurality ofcolumns each column comprising a target receptor or a plurality oftarget receptors, each target receptor optionally attached to a solidphase support, and each column having a inflow end and an outflow end,wherein each of said columns is capable of independently having acompound library comprising a plurality of putative ligands appliedthereto under frontal chromatography conditions to produce an effluentfrom the outflow end of the column; (b) a plurality of first reservoirseach connected to the inflow end of one of the columns for applying acompound library to the columns; (c) a mass spectrometer connected tothe outflow end of each of said columns for intermittently analyzing theeffluent from each of the column.
 15. The apparatus of claim 14 ,wherein said apparatus further comprises: (d) a plurality of secondreservoirs each connected to the inflow end of one of the columns forapplying either (i) a mixture comprising the compound library, at leastone void marker compound and an indicator compound or a plurality ofindicator compounds, (ii) at least one void marker compound and anindicator compound or a plurality of indicator compounds, or (iii) abuffer solution to the column.
 16. The apparatus of claim 14 , whereinsaid apparatus further comprises: (e) a third reservoir connected to theoutflow end of each of the columns for supplying a supplemental diluentto the effluent from each column before analysis by the massspectrometer.
 17. The apparatus of claim 14 , wherein said apparatuscomprises from 2 to about 100 columns.
 18. The apparatus of claim 17 ,wherein said apparatus comprises from 3 to about 50 columns.
 19. Theapparatus of claim 18 , wherein said apparatus comprises from 5 to about10 columns.
 20. The apparatus of claim 14 , wherein each column isintermittently monitored for a period of about 0.5 seconds to about 10seconds before switching to the next column.
 21. The apparatus of claim20 , wherein each column is intermittently monitored for about 1 secondto about 5 seconds before switching to the next column.
 22. Theapparatus of claim 14 , wherein the column has an internal diameterranging from about 10 μm to about 4.6 mm.
 23. The apparatus of claim 22, wherein the column has an internal diameter of from about 100 μm toabout 250 μm.
 24. The apparatus of claim 14 , wherein the column has alength of from about 1 cm to about 30 cm.
 25. The apparatus of claim 14, wherein the column has a length of from about 2 cm to about 20 cm. 26.The apparatus of claim 14 , wherein each target receptor isindependently selected from the group consisting of proteins,glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes,lectins, selectins, cell-adhesion molecules, toxins, bacterial pili,transport proteins, receptors involved in signal transduction orhormone-binding, hormones, antibodies, major histocompatabilitycomplexes, immunoglobulin superfamilies, cadherins, DNA or DNAfragments, RNA and RNA fragments, whole cells, cell fragments, tissues,bacteria, fungi, viruses, parasites, preons, and synthetic analogs orderivatives thereof.
 27. The apparatus of claim 14 , wherein each targetreceptor is bound to a solid phase support.
 28. The apparatus of claim27 , wherein each target receptor is covalently bound to the solid phasesupport or bound via biotin-avidin or biotin-streptavidin binding. 29.The apparatus of claim 27 , wherein the solid phase support is selectedfrom the group consisting of polymeric beads, polymeric gels, glassbeads, silica chips, silica capillaries, agarose, diatomaceous earthsand pulp.
 30. The apparatus of claim 14 , wherein the column containsfrom about 1 fmol to about 10 nmol of target receptor active sites. 31.The apparatus of claim 14 , wherein the mass spectrometer is anelectrospray mass spectrometer.
 32. An apparatus for screening a targetreceptor or a plurality of target receptors to determine the relativeaffinity of the receptor or receptors to an immobilized ligand orligands relative to an indicator compound or a plurality of indicatorcompounds, which apparatus comprises: (a) a column comprising a ligandor a plurality of ligands wherein each ligand is bound to a solid phasesupport, said column having a inflow end and an outflow end and furtherwherein said column is capable of having a target receptor or aplurality of target receptors applied thereto under frontalchromatography conditions to produce an effluent from the outflow end ofthe column; (b) a first reservoir connected to the inflow end of saidcolumn for applying the target receptor or receptors to the column; (c)a second reservoir connected to the inflow end of the column forapplying either (i) a mixture comprising the target receptor orreceptors, at least one void marker compound and an indicator compoundor a plurality of indicators compounds, (ii) at least one void markercompound and an indicator compound or a plurality of indicatorcompounds, or (iii) a buffer solution to the column; (d) a massspectrometer connected to the outflow end of said column forcontinuously or intermittently analyzing the effluent from the column.33. The apparatus of claim 32 , wherein said apparatus furthercomprises: (e) a third reservoir connected to the outflow end of thecolumn for supplying a supplemental diluent to the effluent beforeanalysis by the mass spectrometer.
 34. The apparatus of claim 32 ,wherein the column has an internal diameter ranging from about 10 μm toabout 4.6 mm.
 35. The apparatus of claim 34 , wherein the column has aninternal diameter of from about 100 μm to about 250 μm.
 36. Theapparatus of claim 32 , wherein the column has a length of from about 1cm to about 30 cm.
 37. The apparatus of claim 32 , wherein the columnhas a length of from about 2 cm to about 20 cm.
 38. The apparatus ofclaim 32 , wherein each ligand is selected from the group consisting ofcarbohydrates, monosaccharides, oligosaccharides, polysaccharides, aminoacids, peptides, oligopeptides, polypeptides, proteins, nucleosides,nucleotides, oligonucleotides, polynucleotides, lipids, retinoids,steroids, glycopeptides, glycoproteins, glycolipids, proteoglycans, andsynthetic analogs or derivatives thereof.
 39. The apparatus of claim 32, wherein each ligand is selected from the group consisting of syntheticsmall molecule organic compounds.
 40. An apparatus for screening aplurality of target receptors to determine the relative affinity of thereceptors to an immobilized ligand or ligands relative to an indicatorcompound or a plurality of indicator compounds, which apparatuscomprises: (a) a plurality of columns each column comprising a ligand ora plurality of ligands wherein each ligand is bound to a solid phasesupport, and each column having a inflow end and an outflow end, whereineach of said columns is capable of independently having a targetreceptor or a plurality of target receptors applied thereto underfrontal chromatography conditions to produce an effluent from theoutflow end of the column; (b) a plurality of first reservoirs eachconnected to the inflow end of one of the columns for applying a targetreceptor or a plurality of target receptors to the columns; (c) aplurality of second reservoirs each connected to the inflow end of oneof the columns for applying either (i) a mixture comprising the targetreceptor or plurality of target receptors, at least one void markercompound and an indicator compound or a plurality of indicatorcompounds, (ii) at least one void marker compound and an indicatorcompound or a plurality of indicator compounds, or (iii) a buffersolution to the column; (d) a mass spectrometer connected to the outflowend of each of said columns for intermittently analyzing the effluentfrom each of the column.
 41. The apparatus of claim 40 , wherein saidapparatus further comprises: (e) a third reservoir connected to theoutflow end of each of the columns for supplying a supplemental diluentto the effluent from each column before analysis by the massspectrometer.
 42. The apparatus of claim 40 , wherein said apparatuscomprises from 2 to about 100 columns.
 43. The apparatus of claim 42 ,wherein said apparatus comprises from 3 to about 50 columns.
 44. Theapparatus of claim 43 , wherein said apparatus comprises from 5 to about10 columns.
 45. The apparatus of claim 40 , wherein each column isintermittently monitored for a period of about 0.5 seconds to about 10seconds before switching to the next column.
 46. The apparatus of claim45 , wherein each column is intermittently monitored for about 1 secondto about 5 seconds before switching to the next column.
 47. Theapparatus of claim 40 , wherein the column has an internal diameterranging from about 10 μm to about 4.6 mm.
 48. The apparatus of claim 47, wherein the column has an internal diameter of from about 100 μm toabout 250 μm.
 49. The apparatus of claim 40 , wherein the column has alength of from about 1 cm to about 30 cm.
 50. The apparatus of claim 40, wherein the column has a length of from about 2 cm to about 20 cm. 51.The apparatus of claim 40 , wherein each ligand is selected from thegroup consisting of carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, peptides, oligopeptides, polypeptides,proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides,lipids, retinoids, steroids, glycopeptides, glycoproteins, glycolipids,proteoglycans, and synthetic analogs or derivatives thereof.
 52. Theapparatus of claim 40 , wherein each ligand is selected from the groupconsisting of synthetic small molecule organic compounds.